Bioassays are used to probe for the presence and/or the quantity of an analyte material in a biological sample. In surface based assays, the analyte is quantified by its capture and detection on a solid support. One example of a surface-based assay is a DNA microarray. The use of DNA microarrays has become widely adopted in the study of gene expression and genotyping due to the ability to monitor large numbers of genes simultaneously (Schena et al., Science 270:467-470 (1995); Pollack et al., Nat. Genet. 23:41-46 (1999)). More than 100,000 different probe sequences can be bound to distinct spatial locations across the microarray surface, each spot corresponding to a single gene (Schena et al., Tibtech 16:301-306 (1998)). When a fluorescent-labeled DNA analyte sample is placed over the surface of the array, individual DNA strands hybridize to complementary strands within each array spot. The level of fluorescence detected quantifies the number of copies bound to the array surface and therefore the relative presence of each gene, while the location of each spot determines the gene identity. Using arrays, it is theoretically possible to simultaneously monitor the expression of all genes in the human genome. This is an extremely powerful technique, with applications spanning all areas of genetics. (For some examples, see, the Chipping Forecast supplement to Nature Genetics 21 (1999)). Arrays can also be fabricated using other binding moieties such as antibodies, proteins, haptens or aptamers, in order to facilitate a wide variety of bioassays in array format.
Other surface-based assays include microtitre plate-based ELISAs in which the bottom of each well is coated with a different antibody. A protein sample is then added to each well along with a fluorescent-labeled secondary antibody for each protein. Analyte proteins are captured on the surface of each well and secondarily labeled with a fluorophore. The fluorescence intensity at the bottom of each well is used to quantify the amount of each analyte molecule in the sample. Similarly, antibodies or DNA can be bound to a microsphere such as a polymer bead and assayed as described above. Once again, each of these assay formats is amenable for use with a plurality of binding moieties as described for arrays.
Other bioassays are of use in the fields of proteomics, and the like. For example, cell function, both normal and pathologic, depends, in part, on the genes expressed by the cell (i.e., gene function). Gene expression has both qualitative and quantitative aspects. That is, cells may differ both in terms of the particular genes expressed and in terms of the relative level of expression of the same gene. Differential gene expression is manifested, for example, by differences in the expression of proteins encoded by the gene, or in post-translational modifications of expressed proteins. For example, proteins can be decorated with carbohydrates or phosphate groups, or they can be processed through peptide cleavage. Thus, at the biochemical level, a cell represents a complex mixture of organic biomolecules.
One goal of functional genomics (“proteomics”) is the identification and characterization of organic biomolecules that are differentially expressed between cell types. By comparing expression, one can identify molecules that may be responsible for a particular pathologic activity of a cell. For example, identifying a protein that is expressed in cancer cells but not in normal cells is useful for diagnosis and, ultimately, for drug discovery and treatment of the pathology. Upon completion of the Human Genome Project, all the human genes will have been cloned, sequenced and organized in databases. In this “post-genome” world, the ability to identify differentially expressed proteins will lead, in turn, to the identification of the genes that encode them. Thus, the power of genetics can be brought to bear on problems of cell function.
Differential chemical analyses of gene expression and function require tools that can resolve the complex mixture of molecules in a cell, quantify them and identify them, even when present in trace amounts. The current tools of analytical chemistry for this purpose are presently limited in each of these areas. One popular biomolecular separation method is gel electrophoresis. Frequently, a first separation of proteins by isoelectric focusing in a gel is coupled with a second separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The result is a map that resolves proteins according to the dimensions of isoelectric point (net charge) and size (i.e., mass). Although useful, this method is limited in several ways. First, the method provides information only about two characteristics of a biomolecule-mass and isoelectric point (“pI”). Second, the resolution power in each of the dimensions is limited by the resolving power of the gel. For example, molecules whose mass differ by less than about 5% or less than about 0.5 pI are often difficult to resolve. Third, gels have limited loading capacity, and thus limited sensitivity; one often cannot detect biomolecules that are expressed in small quantities. Fourth, small proteins and peptides with a molecular mass below about 10-20 kDa are not observed.
The use of mass spectrometric methods is replacing gels as the method of choice for bioassays. Efforts to improve the sensitivity of assays have resulted in the application of a number of mass spectrometric formats to the analysis of samples of biological relevance. In addition to the innovations in mass spectrometric techniques, substrates that adsorb an analyte (“chips”) have also developed and the early designs have been improved upon.
Particularly useful methods of performing bioassays rely on the use of an adsorbent chip in conjunction with mass spectrometry. Prior investigators, have reported a variety of techniques for analyte detection using mass spectroscopy, but these techniques suffered because of inherent limitations in sensitivity and selectivity of the techniques, specifically including limitations in detection of analytes in low volume, undifferentiated samples (Hillenkamp, Bordeaux Mass Spectrometry Conference Report, pp. 354-62 (1988); Karas and Hillenkamp, Bordeaux Mass Spectrometry Conference Report, pp. 416-17 (1988); Karas and Hillenkamp, Analytical Chemistry, 60:2299 2301(1988); Karas, et al., Biomed. Environ. Mass Spectrum 18:841-843 (1989)). The use of laser beams in time-of-flight mass spectrometers is shown, for example, in U.S. Pat. Nos. 4,694,167; 4,686,366, 4,295,046, and 5,045,694, incorporated herein by reference.
Exemplary mass spectrometric formats include matrix assisted laser desorption/ionization mass spectrometry (MALDI), see, e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.) and U.S. Pat. No. 5,045,694 (Beavis and Chait), and surface enhanced laser desorption/ionization mass spectrometry (SELDI), see, e.g., U.S. Pat. No. 5,719,060 (Hutchens and Yip), incorporated herein by reference.
Direct laser desorption/ionization of biomolecules, such as polypeptides and nucleic acids, generally results in fragmentation of the biomolecules. To achieve desorption and ionization of intact biomolecules having weights into the hundreds-of-thousands of Daltons, various techniques have been used. In one methodology developed in the 1980's, referred to as MALDI, the biomolecules are mixed in solution with an energy absorbing organic molecule (“EAM”), referred to as a “matrix.” The matrix is allowed to crystallize on a mass spectrometry probe, capturing biomolecules within the matrix. In SELDI, biomolecules are captured by adsorbents bound to a solid phase, and a matrix solution may then be applied to the captured biomolecules. Two very popular matrix materials are sinnapinic acid, which is preferred for use with large biomolecules such as proteins, and cyano hydroxyl cinammic acid, which is preferred for use with peptides and oligonucleotides.
There are a number of problems and limitations with prior matrices. For example, it is difficult to wash away contaminants present in analyte or matrix. Other problems include formation of analyte-salt ion adducts, less than optimum solubility of analyte in matrix, unknown location and concentration of analyte molecules within the solid matrix, signal (molecular ion) suppression “poisoning” due to simultaneous presence of multiple components, and selective analyte desorption/ionization.
Moreover, analysis by means of laser desorption/ionization time-of-flight mass spectrometry requires the preparation of a crystalline solid mixture of the protein or other analyte molecule in a large molar excess of matrix material deposited on the bare surface of a probe. Embedding the analyte in such a matrix is believed to be a necessary condition to prevent the destruction and fragmentation of analyte molecules by a desorption means, e.g., a laser beam. In other words, without the matrix the analyte molecules are easily fragmented by the laser energy and the mass, and identity, of the target macromolecule become very difficult or almost impossible to determine. Proper application of a large amount of matrix molecules over the analyte consistently each time an analysis is performed becomes a cumbersome task for a routine process. Importantly, a small amount of inconsistency in any of the required steps makes an accurate examination of analyte molecules almost impossible.
One notable attempt to overcome the deficiencies of known matrices relied upon chemically modifying the chip by binding small molecular EAM to the surface of the chip. See, for example, U.S. Pat. Nos. 6,027,942; 6,020,208; 6,124,137; and Hutchens and Yip, Tetrahedron Lett. 37: 5669-5672 (1996). The chemically modified chip is disclosed to be advantageous in analyses in which it is desired to modify or derivatize the analyte subsequent to its immobilization on the chip.
The prior methods, relying upon chemical derivatization of the chip substrate with small molecular EAM lacks versatility in a number of regards. For example, attachment of the EAM to the substrate requires the use of EAM and substrate materials having complementary reactive groups, thereby limiting the species that can be used for both the chip and substrate. Moreover, incomplete reaction between EAM and the chip substrate can interfere with the assay for which the chip is intended. For example, unreacted EAM may remain adventitiously, or reactive groups on the surface of the chip may remain unfunctionalized with an EAM. Unreacted EAM may itself be ionized during the mass spectrometric analysis, resulting in a high level of background or obscuring data from the analyte. Unfunctionalized groups on the chip may act as affinity moieties, adventitiously binding the analyte and hindering its desorption from the chip.
A matrix based upon an easily prepared and readily available EAM, that did not require chemical attachment to the substrate is desirable. If the matrix could also be assembled from a wide range of EAM, under a variety of conditions, this would represent a significant advance in the art. The present invention provides such a matrix, chips incorporating the matrix and methods of making and using the matrix.